application of genetic analyzer in aflp technique
DESCRIPTION
the presentation introducing the following points: •Genetic markers. •Restriction Fragment Length Polymorphism (RFLP). •Amplified Fragment Length Polymorphism (AFLP). •Using Genetic Analyzer in AFLP. •Troubleshooting.TRANSCRIPT
Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes Aug. 31st- Sept. 4th, 2014
Application of Genetic Analyzer in AFLP Technique
Amr M. Ageez, Ph.D. Genomics facility, AGERI, ARC,
Giza, Egypt.
• Genetic markers.
• Restriction Fragment Length Polymorphism (RFLP).
• Amplified Fragment Length Polymorphism (AFLP).
• Using Genetic Analyzer in AFLP.
• Troubleshooting.
Contents:
Genetic Marker
• Any phenotypic difference controlled by genes, that
can be used for studying recombination processes or
selection of a more or less closely associated target
gene.
• Anything in the genome that is variable and can be
used to compare individuals.
• Detectable allelic variation on a chromosome can be
a phenotype, can also be a unique detectable
sequence of DNA
• Readily detectable sequence of protein or DNA that are
closely linked to a gene locus and/or a morphological or
other characters of a plant
• Readily detectable sequence of protein or DNA whose
inheritance can be monitored and associated with the
trait inheritance independently from the environment
Molecular Marker
Types:
A) protein polymorphisms
b) DNA polymorphisms
DNA-based markers
• Restriction pattern: RFLPs, minisatellites
• PCR amplicon pattern: RAPDs
• DNA sequence: SNP
• Combinations of above: SCARs, SSRs
(microsatellites), CAPs, AFLP
Restriction Fragment Length Polymorphism (RFLP)
• Genomic DNA digested with Restriction Enzymes
• DNA fragments separated via electrophoresis and
transfer to nylon membrane
• Membranes exposed to probes labelled with P32
via southern hybridization
• Film exposed to X-Ray
RFLP Methodology
(Pawlik, 2008)
Origin of polymorphism in RFLP
According to the lanes:
A: Wild type specific pattern.
B: Insertion.
C: Deletion.
D: New Restriction site within the probe site.
E: Loss of restriction site within the probe site.
Origin of polymorphism in RFLP
(Schuler Group, 2008)
– Reproducible
– Co-dominant
– Simple
Advantages of RFLP Technique
1. Need high quality DNA
2. Need to develop polymorphic probes - expensive
3. Relatively slow process
4. Use of radioisotopes limits use to certified laboratories
(non-radioactive labeling systems now in wide use)
5. Large quantities of DNA are needed and procedure is
difficult to automate
Limitation of RFLP Technique
The AFLP™ technique is used to visualize
hundreds of amplified DNA restriction
fragments simultaneously.
AFLP technology combines the power of
restriction fragment length polymorphism
(RFLP) with the flexibility of PCR-based
technology by ligating primer-recognition
sequences (adaptors) to the restricted
DNA.
AFLP: Amplified Fragment Length Polymorphism
Amplified Fragment Length Polymorphism (AFLP)
• Restriction endonuclease digestion of DNA
• Ligation of adaptors
• Amplification of ligated fragments
• Separation of the amplified fragments viaelectrophoresis and visualization
• AFLPs have stable amplification and goodrepeatability
AFLP …1
Genomic DNA digested with 2 restriction enzymes:– EcoRI (6 bp restriction site)cuts infrequently
– MseI(4 bp restriction site)cuts frequently
GAATTCCTTAAG
TTAAAATT
Fragments of DNA resulting from restriction digestion areligated with end-specific adaptors (a different one for eachenzyme) to create a new PCR priming site
Pre selective PCR amplification is done using primers complementary to the adaptor + 1 bp (chosen by the user)
NN N N
AFLP …2
Selective amplification using primers complementary to the adaptor (+1 bp) + 2 bp
NNNNNN NNN NNN
AFLP …3
Sample AFLP Gel
Advantages of the AFLP:
1. Only small amounts of DNA are needed.
2. Unlike randomly amplified polymorphic DNAs (RAPDs) that use multiple,
arbitrary primers and lead to unreliable results, the AFLP technique uses
only two primers and gives reproducible results.
3. Many restriction fragment subsets can be amplified by changing the
nucleotide extensions on the adaptor sequences. Hundreds of markers
can be generated reliably.
4. High resolution is obtained because of the stringent PCR conditions.
5. The AFLP technique works on a variety of genomic DNA samples.
6. No prior knowledge of the genomic sequence is required.
What is new?
Genomic DNA digested with 2 restriction enzymes:
Adaptors Ligation
Pre selective PCR amplification
NN N N
Selective amplification
NNNNNN NNN NNN
nine EcoRI fluorescent dye-labeled primers and nine unlabeled MseI primers
Machines
PE 310 Single Capillary System
Technology of Capillary Electrophoresis
Technology of Capillary Electrophoresis
Technology of Capillary Electrophoresis
Technology of Capillary Electrophoresis
PE 310 Single Capillary System
Genetic Analyzer 3100 Capillary System
AFLP Electropherogram
Source: Wikimedia Commons
Peak Height
Fragment Size (bp)
AFLP Fluorescent electrophoresis
Tomato AFLP samples showing Mendelian segregation
The overlapping electropherograms in the panel are AFLP results of sample DNA from three
individuals: parent one (P1), parent two (P2), and F1 from a cross. A and B are the two
significant peaks on this panel and appear only in P2 and F1. GeneScan Reference Guide, LifeTech.
Genome size and AFLP
AFLP Core kit
In the microbial genomes targeted by the AFLP Microbial Fingerprinting Kit, the
core primer sequence is used. In larger genomes, such as plants and some fungi,
this amplification would create too many fragments. In those cases, the
preselective amplification is performed with additional nucleotides on the end of
each primer. Each added nucleotide reduces the number of sequences by a
factor of four
• Additional PCR amplifications are run to reduce the
complexity of the mixture further so that the fragments can be
resolved on a polyacrylamide gel. These amplifications use
primers for the selective amplification of the products. After
PCR amplification with these primers, a portion of the
samples is analyzed on a Applied Biosystems DNA
Sequencer
• The sequences of the adaptors and the restriction site serve
as primer binding sites for a subsequent low-level selection
or “preselective” amplification of the restriction fragments.
The MseI complementary primer contains a 3´ C. The EcoRI
complementary primer contains a 3´ A (Regular Plant
Genome Kit modules, lifeTech.) or no base addition (Small
Plant Genome Kit modules, LifeTech.).
examples of AFLP fingerprint patterns that were prepared using different selective
primers. Note that the EcoRI selective primers with one-nucleotide extensions
(EcoRI-A, EcoRI-T, and EcoRI-G) give simpler patterns than that obtained using the
primer with no extra nucleotide (EcoRI-0).GeneScan Reference Guide, LifeTech.
GeneScan Reference Guide, LifeTech.
GeneScan Reference Guide, LifeTech.
unacceptable primer combinations
GeneScan Reference Guide, LifeTech.
unacceptable primer combinations
Troubleshooting
A. Troubleshooting PCR Amplification
Problems with Poor AmplificationFaint or no signal from sample DNA and from positive control
• Insufficient enzyme in reactions
• Incomplete activation of AmpliTaq Gold™ DNA Polymerase
• Too little sample DNA added to reaction
• Incorrect or suboptimal thermal cycler Parameters
• Tubes not seated tightly in the thermal cycler during amplification
• PCR Master Mix not well mixed before Aliquoting
• Primer concentration too low
• Primers degraded
• Too little free Mg2+ in reaction
• Incorrect pH
Troubleshooting
A. Troubleshooting PCR Amplification
Problems with Poor AmplificationGood signal from positive control but faint or no signal from sample DNA
• Sample contains PCR inhibitor (for example, EDTA, or certain dyes)
• Pipetting errors.
• Sample DNA is degraded
• Insufficient sample DNA added because of
• inaccurate quantitation
• Primer choice not optimal (for example, primers may be annealing to sites of
template secondary structure or may have internal secondary structure).
• Tm of primers is lower than expected.
Troubleshooting
A. Troubleshooting PCR Amplification
Problems with Poor AmplificationPoor yield for multiplex PCR
• Non-optimal thermal cycling parameters
• Competition from mispriming and other competing side reactions
• Problems with primer choice, concentration, or degradation
Yield gets progressively poorer for successive PCR amplifications performed over time• Expired or mishandled reagents
Inconsistent yields with control DNA
• Combined reagents not spun to bottom of PCR sample tube.
• Combined reagents left at room temperature or on ice for extended periods of time
• Combined reagents not thoroughly mixed
• Pipetting errors.
Troubleshooting
A. Troubleshooting PCR Amplification
Extra peaks appear with no discernible pattern
• Presence of exogenous DNA
• Nonspecific priming
• Primer-dimer and primer-oligomer artifacts
• Incomplete restriction (and/or ligation if performing AFLP)
• Too much DNA in reaction so that insufficient adaptor is present
• Mixed sample
Troubleshooting
B. Troubleshooting Genetic Analyzer
Data was not automatically analyzed
• Sample Sheet not completed or completed Incorrectly
• Injection List not completed or completed Incorrectly
• Analysis preferences set incorrectly in data collection program
• Insufficient free RAM
Low current
• Small bubble in capillary blocking current flow
• Small bubble in pump block
• Plugged, broken, or no conducting capillary
• Poor quality water in buffer solutions
Troubleshooting
B. Troubleshooting Genetic Analyzer
No current
• Too little or no buffer in anode buffer reservoir
• Electrode bent
• Capillary bent away from electrode
• Unfilled capillary or bubbles in capillary
• Major leaks in system. Polymer does not enter capillary
• Pump blockage (pump is plugged with urea or crystallized buffer)
• Anode buffer valve does not open
• Plugged, broken, or no conducting capillary
• Poor quality water in buffer solutions
• Incorrect polymer solution formulation
• Corrupted firmware
• Syringe Pump Force too low. Capillary is not being filled completely
Troubleshooting
B. Troubleshooting Genetic Analyzer
Current too high
• Decomposition of urea in polymer solution
• Incorrect buffer formulation (most likely too concentrated)
• Arcing to conductive surface on the instrument
Troubleshooting
B. Troubleshooting Genetic Analyzer
Current too high
• Decomposition of urea in polymer solution
• Incorrect buffer formulation (most likely too concentrated)
• Arcing to conductive surface on the instrument
Troubleshooting
B. Troubleshooting Genetic Analyzer
Problems with Peak Resolution
• Poor capillary performance
• Incorrectly prepared and/or old buffer or polymer solutions
• Injection time too long (broad peaks)
• Incorrectly prepared and/or degraded sample
• Incorrect buffer formulation, or polymer composition
• Electrophoresis voltage too high.
• Sample concentrated by evaporation leaving excess salt behind.
• Incomplete strand separation due to insufficient heat denaturation
• Too much DNA in sample
• Poor quality water
• Capillary too short
Troubleshooting
B. Troubleshooting Genetic Analyzer
Problems with Signal Strength and Quality
No signal
• No sample added
• Sample not at bottom of tube
• Air bubble at bottom of sample tube
• Capillary misaligned with electrode
• Capillary bent out of sample tube
• Autosampler not calibrated correctly
• Sealed sample tube septum
Troubleshooting
B. Troubleshooting Genetic Analyzer
Problems with Signal Strength and Quality
Too low signal
• Insufficient sample added
• Samples added to formamide that has degraded to formic acid and formate ions
• Ions in sample
• Sample not thoroughly mixed with formamide/size standard mixture
• Insufficient [F]dNTPs added to PCR reaction
Too high signal
• Too much sample injected into capillary
• Unincorporated [F]dNTPs
Troubleshooting
B. Troubleshooting Genetic Analyzer
Problems with Signal Strength and Quality
High baseline
• Dirty capillary window
• Capillary moved out of position in front of laser window
• Precipitate in polymer
• Incorrectly prepared and/or old buffer or polymer solutions
• Defective capillary
• Matrix made incorrectly resulting in too much correction (also indicated by troughs
under peaks)
Thank you